Amino acids are basic structural units of proteins and peptides. They can also independently act as free amino acids which have various functions, such as controllers of bodily functions or taste components in foods. Therefore, compositional and quantitative analyses of amino acids in samples are extremely important in many technical areas, such as biochemistry, medical care, as well as research and developments of drugs, food and beverages.
FIG. 9 is a flowchart showing a conventional and common procedure of a quantitative analysis of amino acids in a protein.
In the first stage, an acid solution is added to polypeptides which have been produced by breaking down a protein using an appropriate enzyme or another means, after which the polypeptides are heated to break down them to amino acids (Step S51). The obtained mixture of various amino acids is separated according to the kinds of amino acids by an appropriate separation technique, such as liquid chromatography (Step S52). The separated amino acids are individually labelled, and the respective label signals are measured (Step S53). Ultimately, the intensities of the measured label signals are converted into concentrations, for example, by referring to calibration curves prepared beforehand to determine the quantity of each amino acid (Step S54).
Previously, a ninhydrin reaction was used for the labelling of amino acids in Step S53. Currently, a fluorescent dye label is used in many cases. The previously described method for the quantitative analysis of amino acids is an adequately established technique and can yield highly reliable results in quantitative determination. For example, Non-Patent Document 1 discloses a technique for determining the concentration of a protein by the previously described analyzing method. Another example is an amino acid analyzer disclosed in Non-Patent Document 2, in which a measurement corresponding to Step S53 is performed with a mass spectrometer to reduce the processing time as well as improve the measurement accuracy. A technique for breaking down proteins without using the acidic hydrolysis in Step S51 has also been proposed; specifically, the technique simultaneously uses several kinds of protein digestive enzymes (proteases) to completely digest a protein into isolated amino acids.
Thus, various improvements have been made to the amino acid analyzing method according to the procedure shown in FIG. 9, so as to reduce the processing time or improve the measurement accuracy. In any of those variations, the first step is a pretreatment for completely breaking down a sample (i.e. a protein or peptide) into isolated amino acids, and in most cases, acidic hydrolysis is used in this pretreatment.
In the acidic hydrolysis, the break down of polypeptide into amino acids is performed under extreme conditions; i.e. the sample is heated after a high-concentration acid solution is added (as a typical example, the treatment is performed at 110 degrees Celsius for 24 hours, using a 6 mol/L hydrochloric acid solution). Such a dangerous task requires a skilled worker with appropriate experience. Another problem results from the fact that amino acids vary in stability under high temperature; for some kinds of amino acids, the heat reaction time should be short to achieve high recovery efficiency while a long heat reaction time is desired for other kinds of amino acids to achieve high recovery efficiency. Therefore, to exhaustively recover as many kinds of amino acids as possible, it is necessary to perform additional cumbersome tasks, such as dividing the sample into a plurality of lots and changing the heat reaction time for each lot. In some cases, the required heat reaction time may be as long as 72 hours. Thus, the acidic hydrolysis of a protein requires an extremely cumbersome and time-consuming task which can be conducted only by a select group of workers.
The technique of using microwave heating for acidic hydrolysis of polypeptides has also been proposed, which can reduce the heat reaction time. However, such a technique requires a dedicated, non-versatile apparatus for the pretreatment. Furthermore, the handling and operation of such a special apparatus also requires a skilled worker with appropriate experience.
The amino acid analysis which uses acidic hydrolysis for the pretreatment has a more fundamental problem relating to the accuracy of the analysis: An acidic hydrolysis is such a strong chemical reaction that a considerable number of amino acids undergo influences during the reaction of acidic hydrolysis. For example, cysteine becomes structurally unstable due to the reaction of acidic hydrolysis and hence cannot be recovered in a stable quantity. Asparagine is broken down into asparagine acid by acidic hydrolysis, which means that, if asparagine acid has been found in the products of the acidic hydrolysis, it is impossible to determine whether the asparagine acid has originated directly from a peptide or indirectly via asparagine. The same problem also occurs in the case of glutamine, which is broken down into glutamine acid by acidic hydrolysis. Tryptophan is completely broken down by the acidic hydrolysis reaction, and therefore, its quantity cannot be determined. Thus, the quantitative determination is uncertain or virtually impossible for at least six amino acids (cysteine, asparagine, asparagine acid, glutamine, glutamine acid and tryptophan) out of the twenty amino acids which are known as the constituents of proteins. The previously described techniques can still be applied for a sample which contains none of the six amino acids. However, it is inappropriate to use those techniques for a sample which contains any one of the six amino acids or an unknown sample which contains unknown kinds of amino acids.
The method in which the break down of polypeptides is achieved by the use of several kinds of protein digestive enzymes at a time instead of the acidic hydrolysis is free from the aforementioned drawbacks specific to the acidic hydrolysis. However, the biochemical treatment used in the method is extremely complex, and a considerably strict adjustment of the measurement conditions is required to completely digest a polypeptide into isolated amino acids. The adjustment task is so complex and difficult that it needs a worker with select skills and experience.